In clinics, there are often defects and wounds of irregular shapes that need to be filled and repaired. In such cases, the prospects of using injectable scaffolds are very attractive because they can easily fill irregular- shaped defects in situ in a minimally invasive manner. Added benefits are a rapid recovery and improved comfort and satisfaction for the patients. However, current injectable biomaterials have various limitations to clinical applications. We have recently established a strategy to design, synthesize, and fabricate nanofibrous hollow microspheres (NF-HMS) as novel injectable cell carriers for tissue regeneration. NF-HMS are composed entirely of collagen-like nanofibers and have an open and hollow structure purposefully designed to facilitate cell migration, proliferation and tissue regeneration. Our results have indicated that NF-HMS are an excellent injectable biomaterials for tissue regeneration. In the current application, we propose to incorporate bioactive agents (growth factors) into NF-HMS and develop a hierarchical self-assembled injectable scaffolding system for bone regeneration. In this system, bioactive agents will be encapsulated into the biodegradable nanospheres, which are immobilized in the NF-HMS. Cells will adhere, proliferate and migrate on the surface and the inside of the NF-HMS before being injected into defects. Bioactive agents in the nanospheres will be released in a precisely controlled way (by both nanospheres and NF-HMS) to induce cell differentiation and new tissue formation. Our central hypothesis is that this unique injectable system will provide a superior environment for tissue regeneration. For this study, the following specific aims are proposed: 1) To develop injectable hierarchical NF-HMS scaffolding systems with controlled delivery of BMP2. 2) To test this scaffolding system as a promising injectable carrier for bone tissue regeneration. Our long-term goal is to explore and expand the hierarchical NF-HMS scaffolding system to a broad spectrum of biomedical applications. We expect that the outcomes of the proposed research will greatly expand our modality to design a suitable scaffolding system, leading to new advanced regenerative therapies.